Immunomodulatory effects of the polysaccharide from Sinonovacula constricta on RAW264.7 macrophage cells

Abstract This study aimed to evaluate the immunomodulatory effect of the polysaccharide from Sinonovacula constricta (SCP‐1‐1) in RAW264.7 cells. SCP‐1‐1 with a molecular weight of 440.0 kDa consisted of glucose and mannose. The immunomodulatory assay results showed that SCP‐1‐1 could significantly enhance phagocytic ability, NO production, and some cytokines (TNF‐α, IL‐6, and IL‐1β) secretion of RAW264.7 cell in a dose‐dependent manner. Western blot analysis results demonstrated that SCP‐1‐1 could regulate the expression levels of the key proteins in the signaling pathways of RAW264.7 cell and might associated with NF‐κβ and PI3K signaling pathway. These findings could contribute to elucidate the immunomodulatory activities of the polysaccharide from Sinonovacula constricta.

Macrophages are known to contribute to the innate immune response of the host by defending against pathogens infection, cancers, and immunological diseases (Hirayama et al., 2018). Macrophages are activated by mitogen-activated protein kinases (MAPKs), nuclear factor-kappa β (NFκβ), or phosphoinositide-3-kinase (PI3K)/protein kinase B (AKT) signaling pathway and secrete various immunomodulators once the invasion of the external harmful factors (Fang et al., 2017). Furthermore, many polysaccharides demonstrated that they opposed immunomodulatory activity in vitro and in vivo, by activating immune cells, regulating the expressions of pro-inflammatory, and anti-inflammatory cytokines in the lipopolysaccharide (LPS)stimulated RAW264.7 cells (Ramberg et al., 2010;Yin et al., 2019;Zhang et al., 2016). To our knowledge, little research carried out on S. constricta for its bioactive substances. Yuan and et al., (2020) extracted polysaccharides from S. constricta and evaluated its antioxidant activity. However, the information of the immunomodulatory activity of the polysaccharides from S. constricta still be unknown.
In the present study, a novel polysaccharide from S. constricta (SCP-1-1) was isolated and purified. Moreover, the immunomodulatory activity of SCP-1-1 was evaluated using the RAW 264.7 cell model in vitro. This study will enrich our understanding of the structure characteristics and bioactivities of the S. constricta polysaccharide and benefit further investigations into the utilization of similar mollusks.

| Materials and chemicals
Sinonovacula constricta was obtained from the local market (Ningbo, Zhejiang province) in March, 2019. All standard monosaccharides (glucose, mannose, galactose, arabinose, glucose, rhamnose, and xylose), DPPH, and ABTS were obtained from Sigma Chemical Co. (St. Louis MO, USA). Papain (100,000 U/g) was purchased from Shanghai Yuanye Biological Technology Co., Ltd. The other reagents were analytical grade and obtained from Sinopharm Chemical Reagent Co., Ltd.

| Extraction and purification of polysaccharides from Sinonovacula constricta (SCPs)
Sinonovacula constricta was cleaned and separated the shell and the flesh. The flesh was cleaned and smashed with deionized water (1:2, w/v). The flesh homogenate was treated using papain and deionized water (1:10, g/ml) at 50°C for 3 h. After finishing hydrolysis, the hydrolysate was inactivated by heating at 100°C for 5 min and centrifuged at 2,150 × g for 10 min. The protein in the supernatants was removed by Sevag method. The supernatants were concentrated one quarter of the original volume of the solution. Absolute ethanol (3 volumes) added into the concentrated solution, then the mix solution was precipitated at 4°C for 24 h. The precipitate was collected by centrifugation at 1,210 × g for 10 min, following washed twice with absolute ethanol, dissolved in deionized water, and vacuum freeze-dried to obtain a crude S. constricta polysaccharide (SCPs).
The contents of SCPs were determined according to the phenolsulfuric acid method (Dubois et al., 1956) with some modifications, and d-glucose was used as the standard.
The crude SCPs sample (10 mg/ml, 10 ml) was separated and purified with a DEAE agarose gel-FF column (2.6 cm × 30 cm). The elution process was carried out using deionized water and 1.0 M NaCl solution (1.0 ml/min). The main fractions were further purified by Sephacryl S-400 HR (1.6 cm × 60 cm) column. The eluates were collected using an automated collector (BS-100A, Shanghai, Huxi Analytical Instrument Co., Ltd.) and detected using the phenolsulfuric acid method (Dubois et al., 1956;Yuan et al., 2020).
The columns were calibrated with Glucose 180. T-series Dextran (180,2,700,9,750,36,800,133,850, and 2,000,000 Da) were used as the reference compounds. A 20.0 μl aliquot of the sample (1 mg/ml) was injected for each run. Signals were processed online by GPC software package (Agilent Advance Bio SEC, USA) ).

| Monosaccharide composition analysis
The monosaccharide composition of SCPs was analyzed using High-Performance Liquid Chromatography (HPLC). SCPs was firstly hydrolyzed with trifluoroacetic acid at 110°C for 8 h. Then, the monosaccharides of SCP-1-1 were converted into their corresponding acetylated derivatives with PMPs, PMP derivatives were eluted (1 ml/min) by thermo hypersil ODS-2 HPLC columns (250 mm × 4.6 mm) at 25°C. The absorbance rate was monitored at the wavelength of 245 nm . Seven monosaccharides, including glucose, mannose, galactose, arabinose, rhamnose, and xylose were chosen as the standards.

| Fourier transform infrared spectroscopy (FT-IR) analysis
The samples were thoroughly ground with the air-dried KBr (100 mg) powder and pressed into pellets. Fourier transform infrared spectroscopy (FT-IR) spectra were acquired (resolution, 4 cm −1 ) on a Frontier spectrophotometer (PerkinElmer, Shelton, CT, USA) in the vibration range of 400-4,000 cm −1 for three times (Qin et al., 2018).

| Immunomodulatory activity assay
2.4.1 | Cell line and culture RAW 264.7 cells were obtained from the Shanghai Cell Bank of Chinese Academy of Sciences (Shanghai, China) and pre-cultured in an DMEM medium supplemented with 10% (v/v) fetal bovine serum, penicillin-streptomycin solution (100 μg/ml) in a humidified incubator with 5% CO 2 at 37°C. RAW 264.7 cells were cultured and harvested at the logarithmic growth phase.

| Assessment of cell proliferation
The cell viability was determined by CCK-8 method .
The non-adherent cells were removed by washing with PBS (0.1 M, pH 7.2). Then, control and different concentrations of SCPs (100 μl/ well) were added and incubated for 24 h. The DMEM medium without SCPs was chosen as the control group (blank), PBS as the control group (10 μg/ml, negative), LPS as the control group (2 μg/ml, positive), and different concentrations of SCPs, respectively. At the end of culture, the cells were washed twice with PBS, and then 10μl CCK-8 solution (5 mg/ml in the DMEM medium) was added. The plate was further incubated at 37°C for 4 h in a humidified incubator with 5% CO 2 . After the untransformed CCK-8 was removed by pipetting, 150μl DMSO solution/well was added and incubated for 10 min. Cell viability was calculated by the following equation:

| Assay of phagocytosis
The phagocytosis of RAW 264.7 cells was determined by the neutral red staining method (Wang et al., 2017). The cell suspension (1.0 × 10 5 cells/ml in DMEM) was seeded into a 96-well plate (100 μl/well) and allowed in a humidified incubator with 5% CO 2 to adhere at 37°C for 24 h. The non-adherent cells were removed by washing twice with PBS (0.1 M, pH 7.2). Then, different concentrations of SCPs (300, 500, 750, and 1,000 μg/ml, respectively) were added followed by incubation for another 48 h. LPS (2 μg/ ml) and the DMEM medium in the absence of polysaccharide were used as a positive control and a blank control, respectively. At the end of incubation, 20 μl of 0.1% (w/v) neutral red solution (in normal saline) was added and incubated in a humidified incubator with 5% CO 2 at 37°C for 4 h. Following the supernatant was discarded and the cells were washed with PBS twice to remove excess neutral red. The cell lysate (200 μl/well) was added and kept for 10 min. Phagocytosis index was calculated by the following equation: 2.4.4 | Determination of Nitric oxide (NO), TNF-α, IL-6, and IL-1β production NO released into the culture supernatant of RAW 264.7 cells was quantified by measuring the nitrite content. The total NO content was measured by the Griess method . The cell suspension (1.0 × 10 5 cells/ml) was seeded into a 96-well plate (100 μl/well) and incubated at 37 ℃ for 24 h in a humidified incubator with 5% CO 2 . The non-adherent cells were removed by washing twice with PBS buffer (0.1 M, pH 7.2). Then, SCPs (100 μl/well) at different concentrations (300, 500, 750, and 1,000 μg/ml) were, respectively, added into each well followed by incubation for another 24 h. Following, the supernatant of each well was collected for analysis of NO released by RAW 264.7 cells. Nitrite concentration was calculated from the NaNO 2 standard curve (1, 2, 5, 10, 40, 60, and 100 μM, respectively) (Sun, Liu, et al., 2018;Sun, Gong, et al., 2018;Zha et al., 2015). The levels of tumor necrosis factor alpha (TNFα), interleukin 6 (IL-6), and interleukin 1β (IL-1β) were as- Abs (Blank control) × 100 % membrane was blocked using 5% non-fat milk in TBST ((0.1% (v/v) Tween 20, 20 mM Tris-HCl, 150 mM NaCl) for 1 h at 25°C, following incubation at 4°C overnight with the primary antibodies. These membranes were then washed three times (5 min/time) with TBST and incubated with the corresponding secondary antibody at 25°C for 1 h Rong et al., 2019). Immune complexes were visualized by a detection system using an enhanced chemiluminescence kit (Bio-Rad).

| Statistical analysis
All experiments were independently repeated at least three times.
The data were presented as means ± standard deviation (SD). Oneway analysis of variance (ANOVA) with the Duncan's multiple range tests was used for statistical analysis. p < .05 was considered as statistically significant.

| Isolation and purification of SCPs
The yield of crude SCPs was about 0.62% (w/w), but the carbohydrate content of in the crude SCPs by the phenol-sulfuric acid method reached 81.3% (w/w). The crude SCPs by enzymolysis extraction was fractioned by DEAE Sepharose-FF column. The eluent curve of the SCPs is shown in Figure 1. The peaks of two polysaccharide fraction were separated and collected, respectively.
Based on their immunomodulatory activity, SCP-1 was considered over the other fractions and subjected to subsequent investigations. SCP-2 was not further purified because of its lower immunomodulatory activity than that of SCP-1. Then, SCP-1 fraction was further purified using Sephacryl S-400 HR and gained one main sub-fractions (SCP-1-1) ( Figure 2). SCP-1-1 was collected and further analyzed.

| Molecular weight and monosaccharide composition analysis
As high-performance gel-permeation chromatography (HPGPC) is considered a reliable method for measuring the homogeneity of polysaccharides (Xie et al., 2015). The results showed that SCP-1-1 fraction was only a single symmetrical peak and a highly homogeneous polysaccharide with an average molecular weight of about 440.5 kDa. SCP-1-1 was mainly composed of glucose and mannose.
The molecular weight and monosaccharide composition of SCP-1-1 in this study are different from that described by Yuan et al. (2020), which may be due to the difference of the raw material (location, season, etc.), enzyme, and separation protocols.

| Fourier transform infrared spectroscopy (FT-IR) analysis
For the samples, a strong and wide peak at approximately 3,406 cm −1 corresponding to hydrogen-bonded hydroxyl group. An intense peak at    (Feng et al., 2016). These results indicated that SCP-1-1 had no cytotoxic effect on RAW264.7 cells within a certain concentration range (300-1,500 μg/ml).

| Assay of phagocytosis
Phagocytosis is an important defense mechanism against pathogens invasion and dead or expired blood and tissue cells in vertebrates (Dong et al., 2020b). The increase of phagocytosis is the primary and distinguishing feature of macrophage activation (Gordon, 2016).

F I G U R E 3
FT-IR spectra of SCP-1-1 in the range of 4,000-400 cm −1 F I G U R E 4 NMR 1 H-spectrum of SCP-1-1 As shown in Figure 7, the phagocytic capacity increased with the increasing of the concentrations of SCP-1-1. The phagocytosis of RAW264.7 cell in SCP-1-1 groups significantly increased (p < .05) compared with the control group. However, the stimulatory effect of SCP-1-1 (1,000 μg/ml) on the phagocytic rate of macrophages was similar with the LPS group and stronger than that of the control group. The images of fluorescence microscope showed that the fluorescence intensity depending on the macrophages treated with SCP-1-1 or LPS was obviously stronger than that in the control group ( Figure 8). These results indicated that SCP-1-1 could effectively enhance its immunomodulatory effect through moderately promoting the phagocytic activities of macrophages. Wang et al., (2021) also reported that polysaccharide fractions from asparagus (Asparagus officinalis L.) skin had higher immunomodulatory activity.

| Determination of NO, TNF-α, IL-6, and IL-1β
NO is one of the signaling molecules related to macrophage cytolytic function, and is crucial for fighting against microbes, parasites, and tumor cells (Zhang et al., 2020). Cytokines are produced by macrophages and lymphocytes mediate the unleashing of the effective immune response, link the innate and adaptive immunity to induce the necrosis, apoptosis, and acute inflammation responsible for some intracellular signaling events (Huynh et al., 2007). Macrophage activation has become one of the important indicators for improving the body's innate immune system (Hirayama et al., 2018). Thus, macrophage activation is also an important symbol for evaluating on whether polysaccharides have immunomodulatory function. Once macrophages are activated, the secretion of NO and some cytokines (TNFα, IL-6, and IL-1β) are increased. RAW264.7 cells can spontaneously secrete cytokines in the resting state, and such secretion in RAW264.7 cells was significantly promoted by LPS stimulation. The stimulation of LPS simultaneously promoted the production of NO, TNFα, IL-6, and IL-1β of RAW 264.7 cells. The secretion of NO was F I G U R E 5 NMR 13 C-spectrum of SCP-1-1 F I G U R E 6 Effect of SCP-1-1 on the proliferation of RAW264.7 cell. *p < .05 or **p < .01 versus Control; # p < .05 or ## p < .01 versus LPS

| Western blot analysis
NFκβ plays an important role in the immune modulation and inflammatory responses (Ren et al., 2017). Activated NFκβ can promote the secretion of cytokines (TNFα, IL-1β, etc). Thus, the effects of proinflammatory mediators (such as NFκβ) blocked could be considered an effective therapeutic strategy (Janeway & Medzhitov, 2002). The activation of the NFκβ signaling pathway by TLR2 (toll-like receptor 2)/TLR4 (toll-like receptor 4) began as IKK activated by TLR2/TLR4 phosphorylates Iκβα and subsequently Iκβα was degraded (Kawai & Akira, 2017). Western blotting experiments were performed to assay the levels of key proteins in the signaling pathways of RAW264.7 cells to gain further insights on how SCP-1-1 inhibits the growth of cancer cells and promotes apoptosis (Yuan et al., 2013).

| CON CLUS ION
A polysaccharide from Sinonovacula constricta (SCP-1-1) was isolated, purified, and characterized. SCP-1-1 mainly consisted of glucose and mannose with the molecular weight of 440.0 kDa. The immunomodulatory activity results demonstrated that the immunomodulatory activity of SCP-1-1 on RAW264.7 cells was activated by enhancing phagocytic ability of macrophage and promoting NO production and some cytokines (TNFα, IL-6, and IL-1β) secretion. Furthermore, western blot results showed that SCP-1-1 could regulate the expression levels of the key proteins in NFκβ and PI3K signaling pathways.
These results showed that SCP-1-1 could serve as an honest candidate for immunomodulatory purpose for a therapeutical agent or an ingredient of functional foods. The molecular structure of SCP-1-1 F I G U R E 1 0 Effect of SCP-1-1 on the signaling pathways of RAW264.7 cell by Western blot assay. *p < .05 or **p < .01 versus Control; # p < .05 or ## p < .01 versus LPS and its immunomodulatory activity using animal evaluation models in vivo in the future will be focused because this research is a cellbased study.

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L S TATEM ENT
This study does not involve any human or animal testing.

DATA AVA I L A B I L I T Y S TAT E M E N T
The data used to support the findings of this study are available from the corresponding author upon reasonable request.